Probe for mass spectrometry of liquid sample

ABSTRACT

Disclosed is a probe for mass spectrometry of liquid samples, which may effectively ionize the sample without adding a protic solvent to the mobile phase in the ionization method in mass spectrometry of liquid samples. The probe according to the present invention has a structure represented by the Formula [I]:
 
R 2 -A-R 1 [I]
 
(wherein R 1  represents an ionic functional group which becomes an ion in a solvent, R 2  represents a structure which can bind to other substance, and A represents an arbitrary spacer moiety).

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/05961 which has an Internationalfiling date of Jul. 10, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a probe for mass spectrometry of liquidsamples.

BACKGROUND ART

At present, one of the methods by which a sample in a specimen may bequantified with the highest sensitivity and accuracy is massspectrometry. What is important for the measurement of a liquid sampleby mass spectrometry is to select an ionization method suited for thecharacteristics of the compound. For example, when classified based onthe factors by which the compound is made unstable, the ionizationmethods are largely classified into hard ionization and soft ionization.The former includes electron ionization method, chemical ionizationmethod and atmospheric pressure ionization method, and the latterincludes electrospray ionization method, matrix-assisted laserdesorption ionization method and fast atom bombardment ionization.

On the other hand, a method (LC/MS) in which the sample separated byhigh performance liquid chromatography is subjected to mass spectrometryon the same the ionization method suited for the liquid chromatographyis a method for directly ionizing the sample solution flowing out fromthe tip of a capillary, the ionization method is classified into thelatter method in the methods described above. In particular, theapparatus in which an electrospray ionization mass spectrometer and anapparatus for high performance liquid chromatography are connected online is most widely used, and well exhibits its power in identificationof substances indispensable to the body, such as proteins andsaccharides, as well as of environmental hormones. In the electrosprayionization, the possibility that the molecular ion peak of the sample isdetected is extremely low, and the sample is detected as apseudo-molecular ion peak in which sodium is usually attached.

Alternatively, in LC/MS, ionization of the sample is aided by admixing aprotic solvent such as ammonium acetate, formic acid or acetic acid tothe mobile phase.

As a method for aiding ionization in the mass spectrometry of a liquidsample, it is now common to add a protic solvent to the mobile phase ofliquid chromatography in LC/MS. However, the following drawbacks havebeen pointed out: i) when using ammonium acetate in anion mode, the ionof the sample and the ammonium ion are paired, so that the sensitivityis decreased; ii) when using trifluoroacetic acid in cation mode, thesample ion and the trifluoroacetic acid ion are paired, so that thesensitivity is decreased, and in anion mode, ionization is preventedexcept for some cases; and iii) in case of using acetonitrile, an acidmust be added, and in this case, ammonium acetate cannot be used becauseit is not dissolved therein.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide means foreffectively ionizing a sample without adding a protic solvent to themobilize phase in an ionization method in mass spectrometry of a liquidsample.

The present inventors intensively studied to reach the idea that thesample compound may be effectively ionized by using as a probe acompound having, in one molecule, a group which is ionized in a solvent,and a functional group which reacts with a functional group in thesample compound to covalently bind thereto, and by making the probecovalently bind to the sample compound, and experimentally confirmedthat the sample compound ionized by such a probe can be quantified byelectrospray mass spectrometry with high sensitivity, thereby completingthe present invention.

That is, the present invention provides a probe for mass spectrometry ofliquid samples, which is represented by the Formula [I]:R²-A-R¹  [I](wherein R¹ represents an ionic functional group which becomes an ion ina solvent, R² represents a structure which can bind to other substance,and A represents an arbitrary spacer moiety). The present invention alsoprovides a method for mass spectrometry, comprising binding the probeaccording to the present invention to a sample compound in a sampleliquid; and subjecting the obtained bound product to mass spectrometry.The present invention further provides a use of the compound representedby the Formula [I] for the production of a probe for mass spectrometryof liquid samples.

By using the probe according to the present invention, the sample can beeffectively ionized without adding a protic solvent in the mobile phasein the electrospray ionization method, so that electrospray ionizationmass spectrometry can be carried out for various samples with highsensitivity and high accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mass spectrum obtained in the electrospray ionizationmass spectrometry carried out in Example 11 according to the presentinvention.

FIG. 2 shows the mass spectrum obtained in the electrospray ionizationmass spectrometry carried out in Example 11 according to the presentinvention, wherein the probe alone was subjected to the electrosprayionization mass spectrometry.

FIG. 3 schematically shows the constitution of mass spectrometer used inthe mass spectrometry carried out in Example 11.

BEST MODE FOR CARRYING OUT THE INVENTION

The probe for ionization mass spectrometry of liquid samples accordingto the present invention is represented by the above-described Formula[I]. In Formula [I], R¹ represents an ionic functional group whichbecomes an ion in a solvent. R¹ is a group to be ionized in the solvent.R¹ may be any group as long as it is ionized in the solvent used, andmay be either a group to be positively charged or a group to benegatively charged. Examples of R¹ include amines, carboxylic acid andsalts thereof, sulfonic acid and salts thereof, and

(wherein R′, R″ and R′″ represent arbitrary groups which do notadversely affect the present invention, which may be the same ordifferent, preferably hydrogen, halogen, or C₁-C₂₀ linear or branchedalkyl),but R¹ is not restricted to thereto (It should be noted that “halogen”may be any of fluorine, chlorine, bromine and iodine, unless otherwisespecified). Among these, amines are preferred, and especially, theamines represented by the following Formula [II] are preferred

(wherein R³, R⁴ and R⁵ independently represent arbitrary groups which donot adversely affect the present invention, and preferably hydrogen,halogen, or C₁-C₂₀ linear or branched alkyl),

In the above-described Formula [I], R² represents a structure which canbind to other substance. A preferred family of the examples of R² arethe functional groups which can react with the substance so as tocovalently bind to the substance.

In this case, any functional group may be employed as R² as long as itcan react with the substance so as to covalently bind to the substance.Since the liquid samples to be analyzed by mass spectrometry arebiological substances such as proteins and saccharides in most cases, R²is preferably a functional group which reacts with and covalently bindto a functional group that is often included in these substances, thatis, —NH₂, —SH, —COOH, —OH, —CHO and the like. Examples of such afunctional group include SCN—, ClO₂S—,

BrH₂C—, ClOC—, —NH₂, —NHNH₂, —CH₂I, —CH₂ONH₂(—HCl)(which may or may notbe hydrochloric acid salt),

The functional groups (R′) in sample compounds, which react with thesefunctional groups as well as the resulting bonds formed by the reactionsare shown in Table 1 below (In cases where R² is —NH₂, it can bind tothe R² listed in Table 1 as the functional group which may be employedwhen the functional group (R′) in the sample compound is —NH₂).

TABLE 1 Functional Functional Formed Group (R2) Group (R′) Bond SCN——NH₂

ClO₂S— —NH₂

—NH₂

—SH

BrH₂C— —COOH

IH₂C— —COOH

ClOC— —OH

—CHO

—NH₂

—CH₂ONH₂(—HCl)

—NHNH₂ —CHO

As the R², CH₃CH(NH₂)═CH— may also preferably be employed. In this case,2 molecules of the probe react with the sample compound represented byR—CHO as follows:

Additional preferred examples of R² include the groups represented byFormula [VI]:

(wherein X represents halogen),

and represented by Formula [VII]:—(CH₂)_(n)—NH₂  [VII](wherein n represents an integer of 1 to 5).In Formula [VI], the halogen is preferably bromine.

The R² represented by the above-described Formula [VI] reacts withcytosine in a sample compound as follows:

(wherein R represents an arbitrary group such as the sugar moiety of anucleotide or nucleic acid).

Since this R² can bind to cytosine moiety of a sample compound, it canbind to cytidine monophosphate, cytidine diphosphate, cytidinetriphosphate, deoxycytidine monophosphate, deoxycytidine diphosphate anddeoxycytidine triphosphate, as, well as to nucleic acid such as DNA andRNA containing cytosine.

In cases where R² is the above-described

it reacts with guanine in a sample compound and binds thereto asfollows:

(wherein R represents an arbitrary group such as the sugar moiety of anucleotide or nucleic acid).

Since this R² can bind to guanosine monophosphate, guanosinediphosphate, guanosine triphosphate, deoxyguanosine monophosphate,deoxyguanosine diphosphate and deoxyguanosine triphosphate, as well asto nucleic acid such as DNA and RNA containing guanine.

In cases where R² is the group represented by the above-describedFormula [VII], it reacts with phosphate moiety of a sample compound andbind thereto as follows: (In the example below, the case wherein n is 2is shown.)

Although this reaction equation shows the case wherein the samplecompound is deoxyadenosine monophosphate (that is, the base in thenucleotide is adenine and the sugar is deoxyribose), the base may be anybase other than adenine, such as cytosine, guanine, thymine or uracil,and the sugar may be deoxyribose or ribose, because the R² binds tophosphate moiety. Similarly, although the number of phosphate in theabove-described reaction equation is 1, the number of condensedphosphate may be any of 1 to 3 because the R² binds to terminalphosphate. Thus, the R² represented by Formula [VII] can bind to anytypes of nucleoside monophosphate, nucleoside diphosphate and nucleosidetriphosphate. Further, since there exists a free phosphate in nucleicacids, the R² represented by Formula [VII] can bind to nucleic acidssuch as DNA and RNA.

In cases where R² is a group having optical activity, a sample compoundhaving a specific optical activity can be measured by binding the R² tothe sample compound having optical activity. Moreover, the absoluteconfiguration of the optical center in the sample compound can bedetermined.

Preferred examples of R² having optical activity include the groupsrepresented by Formula [VIII]:

(wherein X represents halogen, and R¹⁰ represents C₁-C₅ alkyl).As the halogen, bromine is especially preferred.

The R² represented by Formula [VIII] reacts with an amino acid and bindthereto as follows:

(wherein R represents an arbitrary side chain of the amino acid).

In this case, if the amino acid is a mixture (racemate) of D-isomer andL-isomer, the formed binding products are diastereomers each other. Bycarrying out LC/MS in this state, not only the product can be detectedwith a high sensitivity by a mass spectrometry such as ESI, but also theabsolute configuration of the amino acid can be determined.

The R² is not restricted to the functional groups which can react with afunctional group in the sample compound and covalently bind thereto, butthe groups which bind to the sample compound by intercalation or bycoordinate bond, as well as the groups having cyclic structures thatclathrate the sample compound may also preferably be employed as R².

That is, preferred examples of R² include those having structures whichintercalate into double-stranded nucleic acids. Concrete examples ofsuch R² include the groups represented by Formula [IX] below:

(wherein R¹¹ and R¹² independently represent hydrogen, halogen, C₁-C₅alkyl or C₁-C₅ N,N-dialkylamino).

Since such R² binds to double-stranded nucleic acids by intercalation, aprobe having such R² may preferably be employed for the measurements ofdouble-stranded nucleic acids.

Examples of the groups having cyclic structures that clathrate thesample compound include the groups represented by Formula [X] below:

(wherein R¹³ represents hydroxyl, carboxyl or C₁-C₅ alkyl; and mrepresents an integer of 5 to 9).

The cyclodextrin structure shown by Formula [X]is a cyclicoligosaccharide having a truncated cone structure in which hydroxylgroups face outward and carbon chains face inward. Since the hole in thecyclodextrin is a hydrophobic field, it can clathrate an organicmolecule in the hole utilizing hydrophobic interaction. Utilizing thisphenomenon, a probe having the above-described R² may be used forpurifying water containing various small amounts of organic molecules,and for identifying the substances contained therein. By adding theprobe in an aqueous sample and stirring the mixture, the organicmolecules in the aqueous sample are clathrated in the holes of thecyclodextrin molecules.

Further examples of the groups having cyclic structures which clathratesample compounds include the groups represented by Formula [XI] below.

(wherein R¹⁴ and R¹⁵ independently represent hydrogen, halogen or C₁-C₅alkyl; R¹⁶ represents C₁-C₅ alkyl or C₁-C₅ alkyl which has a carboxylgroup, ester group or an amide group at its terminal; and p representsan integer of 3 to 7).

The calixarene represented by Formula [XI] is a cyclic oligomercomprising benzene rings. The hole encircled by the benzene rings of thecalixarene is a hydrophobic field, it can clathrate an organic moleculein the hole utilizing hydrophobic interaction. Utilizing thisphenomenon, a probe having the above-described R² may be used forpurifying water containing various small amounts of organic molecules,and for identifying the substances contained therein. By adding theprobe in an aqueous sample and stirring the mixture, the organicmolecules in the aqueous sample are clathrated in the holes of thecalixarene.

The group having a cyclic structure which clathrate a sample compound isnot restricted to those described above. For example, a group formed byeliminating one hydrogen atom from crown ether or the like may also beemployed.

Further, as R², the groups obtained by eliminating one hydrogen atomfrom a complex-forming compound such as EDTA, which forms a coordinatebond with the sample compound may also be employed.

In Formula [I], A represents an arbitrary spacer moiety. Since the probeaccording to the present invention binds to the sample compound by R²and ionizes the compound after binding to the sample compound by R¹, theA located between R¹ and R² may have an arbitrary structure. It ispreferred, however, that A comprise a hydrophobic moiety and hydrophilicmoiety because the probe may be used for both hydrophobic solvent andhydrophilic solvent, so that the universality of the probe is increased.Examples of the hydrophobic moiety include aromatic rings such asbenzene ring. Examples of the hydrophilic moiety include structurescontaining ether, amine or ketone. Preferred examples of such A includethose represented by Formula [III] below:

(wherein R⁶ represents C₁-C₂₀ alkylene with the proviso that not lessthan one and not more than half of the —CH₂— units therein maybe-substituted by one or more groups selected from the group consistingof —O—, —CO— and —NH—, and that the alkylene may be substituted by oneor more C₁-C₂₀ alkyl; and Ar represents an aromatic ring which may besubstituted by 1 to 5 C₁-C₆ alkyl).

Preferred examples of the A represented by Formula [III] include thoserepresented by the following Formula [IV]:

(wherein R⁷ may or may not exist, and when it exists, it representsC₁-C₆ alkylene; and R⁸ represents C₁-C₆ alkylene in which an arbitraryhydrogen is substituted by the benzene ring shown in Formula [IV]),or represented by the following Formula [V]:

(wherein R⁷ and R⁸ represent the same meanings as in Formula [IV]; R⁹may or may not exist, and when it exists, it represents C₁-C₆ alkylene).

As the A, those wherein R⁹ is phenylene group in the above-describedFormula [V] may also preferably be employed. This A may especiallypreferably be employed when R² has the structure represented by theabove-described Formula [VI] and R² is

As the A, those represented by —R— may also preferably be employed(wherein R⁶ represents the same meanings as R³ in Formula [III]). This Amay especially preferably be employed when R² is a group having thestructure which intercalates into double-stranded nucleic acids (e.g.,the group represented by the above-described Formula [IX]) and thesample compound is a double-stranded nucleic acid, and when R² is agroup having a cyclic structure which clathrate other substance (e.g.,the group represented by the above-described Formula [X]or [XI]).

As the A, those represented by —R⁶—Ar—R^(6′)— (wherein R⁶ and Arrepresent the, same meanings as in Formula [III]; R^(6′) may or may notexist, and when it exists, it represents the same meanings as the R⁶ inFormula [III] (with the proviso that R⁶ and R^(6′) in the formula may bethe same or different) may also be preferably employed. Preferredexamples of such A include those represented by Formula [XII] below:

(wherein R¹⁷ and R¹⁸ independently represent C₁-C₆ alkylene; and R¹⁹ mayor may not exist, and when it exists, it represents C₁-C₆ alkylene);or represented by the following Formula [XIII]:

(wherein R²⁰ and R²¹ independently represent C₁-C₆ alkylene; and R²² mayor may not exist, and when it exists, it represents C₁-C₆ alkylene).

Although the A may have an arbitrary structure, if the molecular weightof the overall probe is too large, the percentage of the weight of theprobe in the sample compound-probe binding product is large so that thesensitivity of the analysis is decreased. Therefore, the molecularweight of the probe is preferably not more than 1000.

The probe according to the present invention may easily be produced bythose skilled in the art based on known methods. In the Examples below,production processes for producing a plurality of preferred probes areconcretely described.

The reaction between the sample compound and the probe may be carriedout under appropriate conditions based on known technologies dependingon the R² of the probe and the type of the functional group to be boundto R². In the Examples below, conditions for the binding reactionsbetween preferred probes and sample compounds are concretely described.

Examples of the conditions for the respective binding reactions for theabove-described concrete combinations of R² and the functional group(R′) in the sample compound binding to the R² will now be described.However, since these conditions are merely examples and since thefunctional groups can be bound under other conditions, it is apparentfor those skilled in the art that the conditions for the bindingreactions are not restricted to those described below. Further, as forthe R² s not described here, those skilled in the art may easily carryout the binding reactions according to common chemical knowledge.

(1) In cases where R² is SCN— and R′ is —NH₂

To 2 to 3 mg of a sample, 0.2 ml of (ethanol:water:triethylamine 2:2:1v/v) is added and the resulting mixture is evaporated to dryness underreduced pressure. Thereafter, 0.5 ml of(ethanol:water:triethylamine:probe=7:7:1:1 v/v) is added, and themixture is allowed to react at room temperature for 20 minutes. Thesolvent is then evaporated under reduced pressure and the residue isdissolved in an eluent to obtain a sample solution (see B. A.Bidlingmeyer, et al, J. Chromatogr., 336, 93 (1984)).

(2) In cases where R² is ClO₂S— and R′ is —NH₂

To 2 to 3 mg of a sample, 500 μL of 1M aqueous NaHCO₃ solution and 200μL of 1 mg/mL probe solution in acetone are added, and the resultingmixture is heated at 60° C. for 30 minutes. After evaporating theacetone, the residue is dissolved in an eluent to obtain a samplesolution (see Meffin, P. J., et al, J. Pharm. Sci., 66, 583 (1977).

(3) In cases where R² is

and R′ is —NH₂

In 5.0 mL of THF, 1.5 mg of a sample is dissolved and 50 mg of probe isadded, followed by heating the mixture at 60° C. for 30 minutes. Afterevaporating THF, the residue is dissolved in an eluent to obtain asample solution (see Jupill, T. H., Am. Lab., 8 (5), 85-92 (1976)).

(4) In cases where R² is

and R′ is —SH

In 500 μL of water, 2 to 3 mg of a sample is dissolved, and 200 μL of 1mg/mL probe solution in acetonitrile is added, followed by heating theresulting mixture at 60° C. for 30 minutes. The solvent is evaporatedand the residue is dissolved in an eluent to obtain a sample solution(see Nakashima K., et al, Talanta., 32, 167 (1985)).

(5) In cases where R² is BrH₂C— or IH₂C— and R′ is —COOH

In 500 μL of acetone, 2 to 3 mg of a sample is dissolved, and 200 μL of1 mg/mL probe solution in acetone and 1 mg of K₂CO₃ are added, followedby heating the resulting mixture at 60° C. for 30 minutes. The solventis evaporated and the residue is dissolved in an eluent to obtain asample solution (see Dunges, W., Anal. Chem., 49, 442 (1977)).

(6) In cases where R² is ClOC— and R′ is —OH

In 0.5 ml of pyridine, 2 to 3 mg of a sample is dissolved and 0.2 mL of1 mg/mL probe solution in pyridine is added, followed by heating theresulting mixture at 40° C. for 1 hour. The solvent is evaporated andthe residue is dissolved in an eluent to obtain a sample solution (seeSuzuki, A., et al., J. Biochem., 82, 1185 (19773).

(7) In cases where R² is

and R′ is —CHO

To 1.0 ml of methanol, 0.1 ml of acetic acid and 0.2 mL of 1 mg/mL probesolution in methanol, 2 to 3 mg of a sample is added, and the resultingmixture is heated at 40° C. for 30 minutes. The solvent is evaporatedand the obtained residue is dissolved in an eluent to obtain a samplesolution.

(8) In cases where R² is

and R′ is —NH₂

To 0.1 ml of a sample solution in ethanol (10⁻⁴ to 10⁻³M), 1.5 mL of 10mg/mL probe solution in ethanol is added, and the resulting mixture isstirred at room temperature for 5 to 10 minutes. The solvent isevaporated under reduced pressure and the residue is dissolved in aneluent to obtain a sample solution (see Roth, M., Anal. Chem., 43, 880(1971)).

(9) In cases where R² is —CH₂ONH₂. HCl and R′ is

A mixture of 1 to 5 mg of a sample, two drops of triethylamine and 50 mgof a probe is heated at 50° C. for 30 minutes. The solvent is evaporatedunder reduced pressure and the residue is dissolved in an eluent toobtain a sample solution (see Jupille, T. H., Am. Lab., 8 (5), 85-92(1976)).

(10) In cases where R² is —NINH₂ and R′ is —CHO

In 0.5 ml of water, 1 to 5 mg of a sample was dissolved, and 30% aqueousHClO₄ solution in which a probe is dissolved at a concentration of 20mg/mL was added thereto, followed by stirring the resulting mixture atroom temperature for 10 minutes. The solvent is evaporated under reducedpressure and the residue is dissolved in an eluent to obtain a samplesolution (see Newberg, C., et al., Anal. Chim. Acta, 7, 238 (1952)).

(11) In cases where R² is

and R′ is

In 50 μL of phosphate buffer (pH7.0), 0.1 to 5.0 μg of a sample isdissolved, and 200 μL of 1 mg/mL probe solution in phosphate buffer(pH7.0) is added thereto, followed by stirring the resulting mixture atroom temperature for 5 minutes. The solvent is evaporated under reducedpressure and the residue is dissolved in an eluent to obtain a samplesolution.

(12) In cases where R² is

and R′ is

In 50 mL of phosphate buffer (pH 7.0), 0.1 to 5.0 μg of a sample isdissolved, and 200 μL of 1 mg/mL probe solution in phosphate buffer (pH7.0) is added thereto, followed by stirring the resulting mixture atroom temperature for 5 minutes. The solvent is evaporated under reducedpressure and the residue is dissolved in an eluent to obtain a samplesolution.

(13) In cases where R² is —CH₂CH₂NH₂ and R′ is

In 50 μL of phosphate buffer (pH7.0), 0.1 to 5.0 μg of a sample isdissolved, and 200 μL of 1 mg/mL probe solution in phosphate buffer(pH7.0) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added,followed by stirring the resulting mixture at room temperature for 30minutes. The solvent is evaporated under reduced pressure and theresidue is dissolved in an eluent to obtain a sample solution.

The mass spectrometry of a liquid sample using the probe according tothe present invention may be carried out in exactly the same manner asordinary mass spectrometry of a liquid sample after reacting a samplecompound and the probe of the present invention so as to bind the probeto the sample compound. The probe according to the present invention maybe applied to mass spectrometry of any liquid sample, which accompaniesionization of the sample. Preferred examples of the method for massspectrometry of liquid samples include electrospray ionization massspectrometry, atmospheric pressure chemical ionization massspectrometry, thermospray ionization mass spectrometry, particle beammass spectrometry and frit fast atom bombardment ionization massspectrometry, but the method for mass spectrometry is not restrictedthereto. Further, the probe may be applied not only to ordinary massspectrometry, but also to mass spectrometry carried out in an analyzerintegral with a reaction bath. For example, the probe according to thepresent invention may be applied to the mass spectrometry carried out atdownstream of a reaction bath or reaction coil in an analyzer in which areaction bath or reaction coil is incorporated, such as post-column typehigh performance liquid chromatography apparatus or flow injectionanalyzer. In this case, the probe is mixed with the sample at upstreamof the reaction bath or the reaction coil, and the binding reaction maybe carried out in the reaction bath or the reaction coil. The massspectrometry per se may easily be carried out using a commerciallyavailable apparatus, and following the instructions attached to theapparatus.

The term “liquid sample” includes both the sample compound in caseswhere the sample compound to be analyzed is liquid, and the solution ofthe sample compound in cases where the sample compound is solid.

In cases where the probe according to the present invention is used,since the sample compound is surely ionized by binding of the probe tothe sample compound and since the mass of the moiety in the boundproduct is known, which moiety is originated from the probe, the samplecompound may be quantified with high sensitivity and high accuracy byordinary mass spectrometry. The molecular weight of the sample compoundcan be calculated by subtracting the molecular weight of the probe fromthe observed peak value (m/z value).

The present invention will now be described more concretely by way ofexamples thereof. It should be noted that the present invention is notlimited to the following examples.

EXAMPLE 1 Synthesis of Probe (No. 1)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and SCN— as R² was synthesized.

(1) Synthesis of 2-(2-trimethylsiloxyethoxy)-2-phenylethanenitrile

To a 50 ml two-necked flask, 3.0 g (19.9 mmol) of 2-phenyl-1,3-dioxolane(Compound 1), 2.1 ml (21.4 mmol) of trimethylsillyl cyanoide (TMSCN) and0.3 g (0.94 mmol) of ZnI₂ were added and the resulting mixture wasstirred under nitrogen gas flow at room temperature for 2 hours. Afterconfirming the disappearance of the materials by TLC (SiO₂;CH₂Cl₂:n-hexane=1:4 v/v), 100 ml of diethyl ether was added and theresulting mixture was washed with water. The resulting mixture was driedover anhydrous magnesium sulfate and the solvent was evaporated underreduced pressure to obtain a colorless oily product. Identification wascarried out using ¹H-NMR and ESI-TOF mass spectrometry.

Yield: 93% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.12 (s, 9H, Si(CH₃)₃), 3.65-3.84 (m, 4H, —OCH₂CH₂O—), 5.42 (s, 1H, Ar—CH), 7.40-7.58(m, 5H, ArH) ESI-TOF [M+Na]⁺=272

(2) Synthesis of 2-(2-amino-1-phenylethoxy)ethane-1-ol

To a 100 ml two-necked flask, 2.0 g (8.0 mmol) of Compound 2 was addedand the atmosphere was changed to nitrogen after degassing, followed byadding 50 ml of 1M BH₃ solution in THF dropwise for 30 minutes whilecooling the mixture in an ice bath. After stirring the mixture at roomtemperature for 4 hours, disappearance of the materials was confirmed byTLC (SiO₂; n-hexane:ethyl acetate=4:1 v/v). To the mixture, 6N HCl wasadded to make the mixture acidic while cooling the mixture in an icebath and then most of the solvent was evaporated under reduced pressure.After adding aqueous NaOH solution to adjust the pH to 10, the resultingmixture was extracted three times with 100 ml of ethyl acetate and theorganic phase was dried over anhydrous magnesium sulfate. The solventwas evaporated under reduced pressure to obtain a colorless oilyproduct. Identification of the compound was carried out using ¹H-NMR andESI-TOF mass spectrometry.

Yield: 90% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.55 (br s, 3H),2.83 (dd, 1H), 3.15 (dd, 1H), 3.39-3.62 (m, 2H), 3.68-3.78 (m, 2H), 5.00(dd, 1H), 7.40-7.58 (m, 5H) ESI-TOF [M]⁺=181

(3) Synthesis of(tert-butoxy)-N-(2-(2-hydroxyethoxy)-2-phenylethyl)formamide

To a 30 ml two-necked flask, 0.50 g (2.76 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen after degassing. Then 15 mlof THF, 0.28 ml (2.76 mmol) of triethylamine and 0.60 g (2.76 mmol) ofdi-tert-butyl-dicarbonate were added thereto, and the resulting mixturewas stirred at room temperature. Two hours later, disappearance of thematerials was confirmed by TLC (SiO₂; n-hexane:ethyl acetate=1:1), andthen the solvent was evaporated under reduced pressure. The product waspurified by column chromatography (SiO₂; n-hexane:ethyl acetate=1:1) toobtain a colorless oily product. Identification of the compound wascarried out using ¹H-NMR and ESI-TOF mass spectrometry.

Yield: 90% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.37 (s, 9H), 3.10(s, 3H), 3.51-3.64 (m, 4H), 4.33-4.37 (m, 2H), 4.98 (br s, 1H), 5.01(dd, 1H), 7.41-7.58 (m, 5H) ESI-TOF [M+Na]⁺=304

(4) Synthesis of(tert-butoxy)-N-(2-(2-(methylsulfonyloxy)ethoxy)-2-phenylethylformamide

To a 30 ml two-necked flask, 0.50 g (1.78 mmol) of Compound 4 was addedand the atmosphere was changed to nitrogen after degassing. Then 10.0 mlof diethyl ether and 0.23 g (2.31 mmol) of triethylamine were added.While cooling the mixture in an ice bath, 0.22 g (1.96 mmol) ofmethanesulfonyl chloride was added and the resulting mixture was stirredat room temperature. One hour later, disappearance of the materials wasconfirmed by TLC (SiO₂; n-hexane:ethyl acetate=2:1). After evaporatingthe solvent under reduced pressure, the product was purified by columnchromatography (SiO₂; n-hexane:ethyl acetate=2:1) to obtain whitesolids. Identification of the compound was carried out using ¹H-NMR andESI-TOF mass spectrometry.

Yield: 88% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.37 (s, 9H), 3.10(s, 3H), 3.51-3.64 (m, 4H), 4.33-4.37 (m, 2H), 4.98 (br s, 1H), 5.01(dd, 1H), 7.40-7.58 (m, 5H) ESI-TOF [M+Na]⁺=382

(5) Synthesis of(tert-butoxy)-N-(2-(2-iodoethoxy)-2-phenylethyl)formamide

To a 30 ml two-necked flask, 0.50 g (1.39 mmol) of Compound 5 was addedand the atmosphere was changed to nitrogen after degassing. Then 10.0 mlof acetone and 1.10 g (7.25 mmol) of sodium iodide were added and themixture was heated to reflux. Two hours later, disappearance of thematerials was confirmed by TLC (SiO₂; n-hexane:ethyl acetate=1:1), andthe solvent was evaporated under reduced pressure. The product waspurified by column chromatography (SiO₂; n-hexane:ethyl acetate 10:1) toobtain red solids. Identification of the compound was carried out using¹H-NMR and ESI-TOF mass spectrometry.

Yield: 85% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.35 (s, 9H), 3.22(t, 2H), 3.38-3.66 (m, 4H), 5.02 (br s, 1H), 5.06 (dd, 1H), 7.41-7.58(m, 5H) ESI-TOF [M+Na]⁺=414

(6) Synthesis of 2-(2-iodoethoxy)-2-phenylethane isothiocyanate

To a 10 ml two-necked flask, 0.20 g (0.51 mmol) of Compound 6 was addedand the atmosphere was changed to nitrogen after degassing. Then 2.0 mlof methylene chloride and 0.5 ml of trifluoroacetic acid were added andthe resulting mixture was stirred at room temperature. Thirty minuteslater, disappearance of the materials was confirmed by TLC (SiO₂;n-hexane:ethyl acetate 4:1), and the solvent was evaporated underreduced pressure. To the obtained compound, 5.0 ml of a mixed solvent ofmethylene chloride:water=1:1 v/v was added, and then 0.30 g of sodiumhydrogen carbonate and 80.0 μl of thiophosgene were added, followed bystirring the mixture at room temperature. One hour later, disappearanceof the materials was confirmed by TLC (SiO₂; n-hexane:ethylacetate=4:1), and the mixture was extracted three times with 50 ml ofmethylene chloride, followed by drying the organic phase over anhydroussodium sulfate. The solvent was evaporated under reduced pressure toobtain a yellowish orange oily product. Identification of the compoundwas carried out using ¹H-NMR and ESI-TOF mass spectrometry.

Yield: 80% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 3.26-3.33 (m, 2H),3.36-3.79 (m, 2H), 3.80 (dd, 1H), 3.95 (dd, 1H), 5.24 (dd, 1H),7.40-7.58 (in, 5H) ESI-TOF [M+Na]⁺=355

(7) Synthesis of 2-(2-(triethylamino)ethoxy)-2-phenylethaneisothiocyanate iodide

To a 30 ml eggplant type flask, 0.20 g (0.60 mmol) of Compound 7, 5.0 mlof toluene and 1.0 ml of triethylamine were added, and the mixture wasstirred at room temperature. Twenty four hours later, disappearance ofthe materials was confirmed by TLC (SiO₂; n-hexane:ethyl acetate=4:1),and the obtained precipitates were recovered, followed by washing theprecipitates with toluene to obtain yellowish white solids.Identification of the compound was carried out using ¹H-NMR and ESI-TOFmass spectrometry.

Yield: 92% ¹H-NMR (300 MHz, DMSO-d₆, TMS, r.t., δ/ppm) 1.30 (t, 9H),3.04 (q; 6H), 3.75-3.95 (m, 3H), 4.11 (dd, 1H), 4.104.16 (m, 1H), 4.33(dd, 1H), 5.54 (dd, 1H), 7.40-7.58 (m, 5H) ESI-TOF [M]⁺=307

EXAMPLE 2 Synthesis of Probe (No. 2)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and BrH₂C— as R² was synthesized.

(1) Synthesis of2-(3,3,4,4-tetramethyl-1-phenyl-3-silapentyloxy)ethane-1-ol

To a 50 ml two-necked flask, 3.0 g (16.5 mmol) of2-(2-hydroxyethoxy)-2-phenylethane-1-ol (Compound 1), 2.48 g (16.5 mmol)of t-butyldimethylsilyl chloride (TBDMSCl) and 2.51 ml (16.5 mmol) of1,8-diazabicyclo [5,4,0]-7-undecene (DBU) were added, and the mixturewas stirred under nitrogen gas flow at room temperature for 24 hours.After confirming the disappearance of the materials by TLC, the solventwas evaporated under reduced pressure and the obtained compound wasdissolved in methylene chloride. The solution was washed with water andthen dried over anhydrous magnesium sulfate. The solvent was evaporatedunder reduced pressure and the residue was purified by silica gel columnchromatography to obtain the desired product.

(2) Synthesis of (methylsulfonyl)oxy(2-(3,3,4,4-tetramethyl-1-phenyl-3-silapentyloxy)ethane

To a 100 ml two-necked flask, 2.0 g (6.75 mmol) of Compound 2 was addedand the atmosphere was changed to nitrogen after degassing. Then 20.0 mlof diethyl ether, 0.68 g (6.75 mmol) of triethylamine and 0.77 g (6.75mmol) of methanesulfonyl chloride were added, and the mixture wasstirred at room temperature for 2 hours. The mixture was washed withwater and then dried over anhydrous magnesium sulfate. The solvent wasevaporated under reduced pressure and the residue was purified by silicagel chromatography to obtain the desired compound.

(3) Synthesis of2-iodo-1-(3,3,4,4-tetramethyl-1-phenyl-3-silapentyloxy)ethane

To a 30 ml two-necked flask, 0.50 g (1.25 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen after degassing. Then 15 mlof acetone and 1.49 g (10.0 mmol) of sodium iodide were added and themixture was heated to reflux for 2 hours. After evaporating the solventunder reduced pressure, the residue was washed with water and dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure and the residue was purified by silica gel columnchromatography to obtain the desired compound.

(4) Synthesis of triethyl(2-(3,3,4,4-tetramethyl-1-phenyl-3-silapentyloxy)ethyl)amine

To a 30 ml eggplant type flask, 0.50 g (1.25 mmol) of Compound 4, 10.0ml of toluene and 1.0 ml of triethylamine were added, and the mixturewas stirred at room temperature for 24 hours. The obtained precipitateswere recovered and washed with toluene, followed by drying under reducedpressure to obtain the desired compound.

(5) Synthesis of1-(methylsulfonyl)-2-phenyl-2-(2-(triethylamino)ethoxy)ethane

To a 30 ml two-necked flask, 0.50 g (1.37 mmol) of Compound 5 and 10.0ml of a mixed solvent of acetic acid:water:THF=3:1:1 v/v were added andthe mixture was stirred at room temperature for 2 hours. The solvent wasevaporated under reduced pressure and the resultant was dried underreduced pressure using a pump. To the obtained compound, 10.0 ml ofdiethyl ether, triethylamine and methanesulfonyl chloride were added andthe resulting mixture was stirred at room temperature for 2 hours. Themixture was washed with water and then dried over anhydrous magnesiumsulfate. The solvent was evaporated and the residue was purified bysilica gel column chromatography to obtain the obtained compound.

(6) Synthesis of (2-(2-iodo-1-phenylethoxy)ethyl)triethylamine

To a 50 ml eggplant type flask, 0.20 g (0.58 mmol) of Compound 6, 20.0ml of acetone and 0.43 g (5.0 mmol) of lithium bromide were added andthe mixture was heated to reflux for 2 hours. The solvent was evaporatedunder reduced pressure and ethanol was added. Insoluble materials wereremoved by filtration and the filtrate was concentrated under reducedpressure to obtain the desired compound.

EXAMPLE 3 Synthesis of Probe (No. 3)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and NH₂— as R² was synthesized.

(1) Synthesis of 2-(2-trimethylsiloxyethoxy)-2-phenylethanenitrile

To a 50 ml two-necked flask, 3.0 g (19.9 mmol) of 2-phenyl-1,3-dioxolane(Compound 1), 2.1 ml (21.4 mmol) of TMSCN and 0.3 g (0.94 mmol) of ZnI₂were added and the mixture was stirred under nitrogen gas flow at roomtemperature for 2 hours. After confirming the disappearance of thematerials by TLC (SiO₂; CH₂Cl₂:n-hexane=1:4 v/v), 100 ml of diethylether was added and the resulting mixture was washed with water. Themixture was dried over anhydrous magnesium sulfate and the solvent wasevaporated under reduced pressure to obtain a colorless oily compound.Identification of the compound was carried out using ¹H-NMR and ESI-TOFmass spectrometry.

Yield: 93% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.12 (s, 9H, Si(CH₃)₃), 3.65-3.84 (m, 4H, —OCH2CH₂O—), 5.42 (s, 1H, Ar—CH), 7.40-7.58(m, 5H, ArH) ESI-TOF [M+Na]⁺=272

(2) Synthesis of 2-(2-amino-1-phenylethoxy)ethane-1-ol

To a 100 ml two-necked flask, 2.0 g (8.0 mmol) of Compound 2 was addedand the atmosphere was changed to nitrogen after degassing, followed byadding 50 ml of 1M BH₃ solution in THF dropwise for 30 minutes whilecooling the mixture in an ice bath. After stirring the mixture at roomtemperature for 4 hours, disappearance of the materials was confirmed byTLC (SiO₂; n-hexane:ethyl acetate=4:1 v/v). To the mixture, 6N HCl wasadded to make the mixture acidic while cooling the mixture in an icebath and then most of the solvent was evaporated under reduced pressure.After adding aqueous NaOH solution to adjust the pH to 10, the resultingmixture was extracted three times with 100 ml of ethyl acetate and theorganic phase was dried over anhydrous magnesium sulfate. The solventwas evaporated under reduced pressure to obtain a colorless oilyproduct. Identification of the compound was carried out using ¹H-NMR andESI-TOF mass spectrometry.

Yield: 90% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.55 (br s, 3H),2.83 (dd, 1H), 3.15 (dd, 1H), 3.39-3.62 (m, 2H), 3.68-3.78 (m, 2H), 5.00(dd, 1H), 7.40-7.58 (m, 5H) ESI-TOF [M]⁺=181

(3) Synthesis of(tert-butoxy)-N-(2-(2-hydroxyethoxy)-2-phenylethyl)formamide

To a 30 ml two-necked flask, 0.50 g (2.76 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen after degassing. Then 15 mlof THF, 0.28 ml (2.76 mmol) of triethylamine, 0.60 g (2.76 mmol) ofdi-tert-butyl-dicarbonate were added thereto, and the resulting mixturewas stirred at room temperature. Two hours later, disappearance of thematerials was confirmed by TLC (SiO₂; n-hexane:ethyl acetate=1:1), andthen the solvent was evaporated under reduced pressure. The product waspurified by column chromatography (SiO₂; n-hexane:ethyl acetate=1:1) toobtain a colorless oily product. Identification of the compound wascarried out using ¹H-NMR and ESI-TOF mass spectrometry.

Yield: 90% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.37 (s, 9H), 3.10(s, 3H), 3.51-3.64 (m, 4H), 4.33-4.37 (m, 2H), 4.98 (br s, 1H), 5.01(dd, 1H), 7.41-7.58 (m, 5H) ESI-TOF [M+Na]⁺=304

(4) Synthesis of(tert-butoxy)-N-(2-(2-(methylsulfonyloxy)ethoxy)-2-phenylethylformamide

To a 30 ml two-necked flask, 0.50 g (1.78 mmol) of Compound 4 was addedand the atmosphere was changed to nitrogen after degassing. Then 10.0 mlof diethyl ether and 0.23 g (2.31 mmol) of triethylamine were added.While cooling the mixture in an ice bath, 0.22 g (1.96 mmol) ofmethanesulfonyl chloride was added and the resulting mixture was stirredat room temperature. One hour later, disappearance of the materials wasconfirmed by TLC (SiO₂; n-hexane:ethyl acetate=2:1). After evaporatingthe solvent under reduced pressure, the product was purified by columnchromatography (SiO₂; n-hexane:ethyl acetate=2:1) to obtain whitesolids. Identification of the compound was carried out using ¹H-NMR andESI-TOF mass spectrometry.

Yield: 88% ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.37 (s, 9H), 3.10(s, 3H), 3.51-3.64 (m, 4H), 4.33-4.37 (m, 2H), 4.98 (br s, 1H), 5.01(dd, 1H), 7.40-7.58 (m, 5H) ESI-TOF [M+Na]⁺=382

(5) Synthesis of(tert-butoxy)-N-(2-(2-iodoethoxy)-2-phenylethyl)formamide

To a 30 ml two-necked flask, 0.50 g (1.39 mmol) of Compound 5 was addedand the atmosphere was changed to nitrogen after degassing. Then 10.0 mlof acetone and 1.10 g (7.25 mmol) of sodium iodide were added and themixture was heated to reflux. Two hours later, disappearance of thematerials was confirmed by TLC (SiO₂; n-hexahe:ethyl acetate 1:1), andthe solvent was evaporated under reduced pressure. The product waspurified by column chromatography (SiO₂; n-hexane:ethyl acetate=10:1) toobtain red solids. Identification of the compound was carried out using¹H-NMR and ESI-TOF mass spectrometry.

Yield: 85%. ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.35 (s, 9H), 3.22(t, 2H), 3.38-3.66 (m, 4H), 5.02 (br s, 1H), 5.06 (dd, 1H), 7.41-7.58(m, 5H) ESI-TOF [M+Na]⁺=414

(6) Synthesis of N-(2-(2-iodoethoxy)-2-phenylethyl)-3-oxobutaneamide

To a 10 ml two-necked flask, 0.20 g (0.51 mmol) of Compound 6 was addedand the atmosphere was changed to nitrogen after degassing. Then 2.0 mlof methylene chloride and 0.5 ml of trifluoroacetic acid were added, andthe mixture was stirred at room temperature. Thirty minutes later,disappearance of the materials was confirmed by TLC (SiO₂;n-hexane:ethyl acetate=4:1), and the solvent was evaporated underreduced pressure. To the obtained compound, 5.0 ml of methylenechloride, 0.05 g (0.51 mmol) of trimethylamrine and 0.05 g (0.51 mmol)of acetoacetic acid were added, and the mixture was stirred for 30minutes while cooling the mixture in an ice bath. After adding 0.22 g(0.51 mmol) of BOP(Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate) reagent, the mixture was stirred for 30 minuteswhile cooling the mixture in an ice bath and then for 24 hours at roomtemperature. Disappearance of the materials was confirmed by TLC (SiO₂;n-hexane:ethyl acetate=4:1), and the mixture was extracted three timeswith 50 ml of methylene chloride and the organic phase was dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure to obtain the compound.

(7) Synthesis of3-amino-N-(2-(2-iodoethoxy)-2-phenylethyl)-2-buteneamide

To a 10 ml eggplant type flask, 0.20 g (0.60 mmol) of Compound 7, 5.0 mlof aqueous ammonia and 0.1 g of montmorillonite K-10 were added, and themixture was stirred for 24 hours at room temperature. After extractingthe mixture with methylene chloride, the organic phase was washed withwater and dried over anhydrous magnesium sulfate. The solvent wasevaporated under reduced pressure and the residue was purified by silicagel column chromatography.

(8) Synthesis of3-amino-N-(2-phenyl-2-(2-(triethylamino)ethoxy)ethyl)-2-buteneamide

To a 10 ml eggplant type flask, 0.20 g (0.58 mmol) of Compound 8, 5.0 mlof toluene and 1.0 ml of triethylamine were added, and the mixture wasstirred at room temperature for 24 hours. The generated precipitateswere recovered and washed with toluene, followed by drying under reducedpressure.

EXAMPLE 4 Synthesis of Probe (No. 4)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and —COCH₂Br as R² was synthesized.

(1) Synthesis of phenyl-(2-trimethylsilanyloxy-ethoxy)-acetonitrile(Compound 2)

To a 30 ml eggplant type flask, 3.0 g (19.9 mmol) of2-phenyl-1,3-pentadione (Compound 1) was added and the flask was dippedin an ice bath. To the flask, 2.1 ml (21.43 mmol) of TMSCN and 0.3 g(0.94 mmol) of ZnI₂ were added, and the mixture was stirred at roomtemperature for 2 hours. Diethyl ether was added to the reactionsolution and the mixture was washed with water, followed by drying usingMgSO₄. The solvent was evaporated under reduced pressure to obtain ayellow oily product (4.17 g, yield: 83.2%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.15(s, 9H, SiCH₃), 3.80(t,2H, —OCH₂CH₂OSi—), 4.11(t, 2H, —OCH₂CH₂OSi—), 5.39(s, 1H, ARCH), 7.45(m,5H, ArH)ESI-TOF(+): [M+Na]⁺272.0

(2) Synthesis of 2-(2-amino-1-phenyl-ethoxy)-ethanol (Compound 3)

To a 100 ml three-necked flask, 1.0 g (4.01 mmol) of Compound 2 wasadded and the atmosphere was changed to nitrogen after degassing. Theflask was-dipped in an ice bath and 30 ml of 1M BH₃ solution in THF wasslowly added. The resulting mixture was stirred for 30 minutes whilecooling the mixture in ice and then at room temperature for 4 hours.After completion of the reaction, the reaction vessel was dipped in anice bath and aqueous 1N HCl solution was added, thereby making themixture acidic. After evaporating the solvent under reduced pressure, 20ml of water was added and aqueous NaOH solution was added to adjust thepH to 10. The mixture was extracted with ethyl acetate and the organicphase was washed with water, followed by drying over anhydrous sodiumsulfate to obtain a colorless oily compound (650 mg, Yield: 89.5,%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.95(d, 2H, NH₂—CH₂), 3.65(t,2H, —OCH₂CH₂OH), 3.75(t, 2H, —OCH₂CH₂OH), 4.46(t, 1H, ARCH), 7.32(m, 5H,ArH)ESI-TOF (+): [M+H]⁺=182.0

(3) Synthesis of [2-(2-hydroxy-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester

To a 100 ml two-necked flask, 1.40 g (7.70 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen. While cooling the flask inan ice bath, 55 ml of anhydrous THF, 0.78 g (7.69 mmol) of TEA and 1.68g (7.70 mmol) of di-tert-butyl-dicarbonate were added, and the mixturewas stirred at room temperature for 2 hours. The solvent was evaporatedunder reduced pressure and the residue was purified by columnchromatography (SiO₂, chloroform) to obtain a yellow oily compound (1.30g, Yield: 59.9%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.48(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₃), 3.49(t, 2H, —OCH₂CH₂OH), 3.73(t, 2H, —OCH₂CH₂OH), 4.42(t, 1H,ArCH), 7.33(m, 5H, ArH) ESI-TOF (+): [M+Na]⁺=304.0

(4) Synthesis of methanesulfonic acid2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy) ethyl ester (Compound 5)

To a 50 ml eggplant type flask, 500 mg (1.78 mmol) of Compound 4 wasadded and the atmosphere was changed to nitrogen. Then 16 ml ofanhydrous THF and 0.5 ml (4.55 mmol) of TEA were added and the flask wasdipped in an ice bath. To the mixture, 440 mg (3.84 mmol) of MsCl wasadded and the resulting mixture was stirred at room temperature for 1hour. After evaporating the solvent under reduced pressure, chloroformwas added and the generated precipitates were removed by filtration,followed by concentrating the filtrate under reduced pressure. Theresulting product was purified by column chromatography (SiO₂, ethylacetate: n-hexane=2:1 v/v) to obtain a yellow oily product (491 mg,Yield: 77.0%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.44(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₂), 3.44(t, 2H, —OCH₂CH₂OS—), 3.62(t, 2H, —OCH₂CH₂OS—), 4.34(t, 1H,ArCH), 7.33(m, 5H, ArH) ESI-TOF (+): [M+Na]⁺=382.2

(5) Synthesis of [2-(2-iodo-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester (Compound-6)

To a 50 ml eggplant type flask, 100 mg (0.28 mmol) of Compound 5 wasadded and the atmosphere was changed to nitrogen. Then 10 ml of acetoneand 10.0 g (6.67 mmol) of NaI were added, and the mixture was heated toreflux for 2 hours. After removing NaI by filtration, the solvent wasevaporated under reduced pressure. The residue was purified by columnchromatography (SiO₂, n-hexane:ethyl acetate=1:1 v/v) to obtain a yellowoily compound (104 mg, Yield: 93.99%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.45(s, 9H, t-Bu), 3.22(t, 2H,I—CH₂), 3.49(d, 2H, NH—CH₂), 3.67(t, 2H, —OCH₂), 4.43(t, 1H, ArCH),7.34(m, 5H, ArH)ESI-TOF (+): [M+Na]⁺=413.9

(6) Synthesis of[2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy)-ethyl]-triethyl-ammoniumiodide (Compound 2)

To a 50 ml eggplant type flask, 350 mg (0.90 mmol) of Compound 6, 8.75ml of toluene and 1.75 ml (17.33 mmol) of TEA were added, and thereaction was carried out at 80° C. for 24 hours. After evaporating thesolvent under reduced pressure, the residue was purified by large thinlayer chromatography (SiO₂, chloroform:methanol=7:1 v/v) to obtain areddish yellow solid product (383 mg,

Yield: 66.70%). ¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.42(t, 9H,CH₂—CH₃), 1.48(s, 9H, t-Bu), 3.53(q, 6H, NR₃—CH₂), 3.70(t, 2H,—OCH₂CH₂N—), 3.72(d, 2H, NH—CH₂), 3.95(t, 2H, —OCH₂CH₂N—), 4.63(t, 1H,ArCH), 7.35(m, 5H, ArH) ESI-TOF (+): [M]⁺365.2

(7) Synthesis of Compound 8

To a 30 ml eggplant type flask, 0.50 g (1.02 mmol) of Compound 7, 0.50ml of TFA (trifluoroacetic acid) and 10.0 ml of methylene chloride wereadded, and the mixture was stirred at room temperature for 30-minutes.The solvent was evaporated under reduced pressure and the resultant wasdried under reduced pressure using a pump. After changing the atmosphereto nitrogen, 20.0 ml of THF, 0.10 g (1.02 mmol) of TEA, 0.10 g (1.02mmol) of BOP and 0.25 g (1.02 mmol) of 4-bromomethylacetyl benzoic acidwere added, and the mixture was stirred at room temperature for 24hours. After evaporating the solvent under reduced pressure, the residuewas purified by column chromatography to obtain the desired compound.

EXAMPLE 5 Synthesis of Probe (No. 5)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and —COCHO as R² was synthesized.

(1) Synthesis of phenyl-(2-trimethylsilanyloxy-ethoxy)-acetonitrile(Compound 2)

To a 30 ml eggplant type flask, 3.0 g (19.9 mmol) of2-phenyl-1,3-pentadione (Compound 1) was added and the flask was dippedin an ice bath. To the flask, 2.1 ml (21.43 mmol) of TMSCN and 0.3 g(0.94 mmol) of ZnI₂ were added, and the mixture was stirred at roomtemperature for 2 hours. Diethyl ether was added to the reactionsolution and the mixture was washed with water, followed by drying usingMgSO₄. The solvent was evaporated under reduced pressure to obtain ayellow oily product (4.17 g, yield: 83.2%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.15(s, 9H, SiCH₃), 3.80(t,2H, —OCH₂CH₂OSi—), 4.11(t, 2H, —OCH₂CH₂OSi—), 5.39(s, 1H, ArCH), 7.45(m,5H, ArH)ESI-TOF (+): [M+Na]⁺=272.0

(2) Synthesis of 2-(2-amino-1-phenyl-ethoxy)-ethanol (Compound 3)

To a 100 ml three-necked flask, 1.0 g (4.01 mmol) of Compound 2 wasadded and the atmosphere was changed to nitrogen after degassing. Theflask was dipped in an ice bath and 30 ml of 1M BH₃ solution in THF wasslowly added. The resulting mixture was stirred for 30 minutes whilecooling the mixture in ice and then at room temperature for 4 hours.After completion of the reaction, the reaction vessel was dipped in anice bath and aqueous 1N HCl solution was added, thereby making themixture acidic. After evaporating the solvent under reduced pressure, 20ml of water was added and aqueous NaOH solution was added to adjust thepH to 10. The mixture was extracted with ethyl acetate and the organicphase was washed with water, followed by drying over anhydrous sodiumsulfate to obtain a colorless oily compound (650 mg, Yield: 89.5%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.95(d, 2H, NH₂—CH₂), 3.65(t,2H, —OCH₂CH₂OH), 3.75(t, 2H, —OCH₂CH₂OH), 4.46(t, 1H, ArCH), 7.32(m, 5H,ArH)

ESI-TOF (+): [M+H]⁺=182.0

(3) Synthesis of [2-(2-hydroxy-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester (Compound 4)

To a 100 ml two-necked flask, 1.40 g (7.70 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen. While cooling the flask inan ice bath, 55 ml of anhydrous THF, 0.78 g (7.69 mmol) of TEA and 1.68g (7.70 mmol) of di-tert-butyl-dicarbonate were added, and the mixturewas stirred at room temperature for 2 hours. The solvent was evaporatedunder reduced pressure and the residue was purified by columnchromatography (SiO₂, chloroform) to obtain a yellow oily, compound(1.30 g, Yield: 59.9%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.48(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₃), 3.49(t, 2H, —OCH₂CH₂OH), 3.73(t, 2H, —OCH₂CH₂OH), 4.42(t, 1H,ArCH), 7.33(m, 5H, ArH)

ESI-TOF (+): [M+Na]⁺=304.0

(4) Synthesis of methanesulfonic acid2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy) ethyl ester (Compound 5)

To a 50 ml eggplant type flask, 500 mg (1.78 mmol) of Compound 4 wasadded and the atmosphere was changed to nitrogen. Then 16 ml ofanhydrous THF and 0.5 ml (4.55 mmol) of TEA were added and the flask wasdipped in an ice bath. To the mixture, 440 mg (3.84 mmol) of MsCl wasadded and the resulting mixture was stirred at room temperature for 1hour. After evaporating the solvent under reduced pressure, chloroformwas added and the generated precipitates were removed by filtration,followed by concentrating the filtrate under reduced pressure. Theresulting product was purified by column chromatography (SiO₂, ethylacetate: n-hexane=2:1 v/v) to obtain a yellow oily product (491 mg,Yield: 77.0%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.44(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₂), 3.44(t, 2H, —OCH₂CH₂OS—), 3.62(t, 2H, —OCH₂CH₂OS—), 4.34(t, 1H,ArCH), 7.33(m, 5H, ArH)

ESI-TOF (+): [M+Na]⁺382.2

(5) Synthesis of [2-(2-iodo-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester (Compound 6)

To a 50 ml eggplant type flask, 100 mg (0.28 mmol) of Compound 5 wasadded and the atmosphere was changed to nitrogen. Then 10 ml of acetoneand 1.0 g (6.67 mmol) of NaI were added, and the mixture was heated toreflux for 2 hours. After removing NaI by filtration, the solvent wasevaporated under reduced pressure. The residue was purified by columnchromatography (SiO₂, n-hexane:ethyl acetate=1:1 v/v) to obtain a yellowoily compound (104 mg, Yield: 93.99%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.45(s, 9H, t-Bu) 3.22(t, 2H,I—CH₂), 3.49(d, 2H, NH—CH₂), 3.67(t, 2H, —OCH₂), 4.43(t, 1H, ArCH),7.34(m, 5H, ArH)ESI-TOF (+): [M+Na]⁺413.9

(6) Synthesis of[2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy)-ethyl]-triethyl-ammoniumiodide (Compound 7)

To a 50 ml eggplant type flask, 350 mg (0.90 mmol) of Compound 6, 8.75ml of toluene and 1.75 ml (17.33 mmol) of TEA were added, and thereaction was carried out at 80° C. for 24 hours. After evaporating thesolvent under reduced pressure, the residue-was purified by large thinlayer chromatography (SiO₂, chloroform:methanol 7:1 v/v) to obtain areddish yellow solid product (383 mg, Yield: 66.70%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppmr) 1.42(t, 9H, CH₂—CH₃), 1.48(s,9H, t-Bu), 3.53(q, 6H, NR₃—CH₂), 3.70(t, 2H, —OCH₂CH₂N—), 3.72(d, 2H,NH—CH₂), 3.95(t, 2H, —OCH₂CH₂N—), 4.63(t, 1H, ArCH), 7.35(m, 5H, ArH)ESI-TOF (+): [M]⁺=365.2

(7) Synthesis of Compound 8

To a 30 ml eggplant type flask, 0.50 g (1.02 mmol) of Compound 7, 0.50ml of TFA (trifluoroacetic acid) and 10.0 ml of methylene chloride wereadded, and the mixture was stirred at room temperature for 30 minutes.The solvent was evaporated under reduced pressure and the resultant wasdried under reduced pressure using a pump. After changing the atmosphereto nitrogen, 20.0 ml of THF, 0.10 g (1.02 mmol) of TEA, 0.10 g (1.02mmol) of BOP and 0.25 g (1.02 mmol) of 4-bromomethylacetylbenzoic acidwere added, and the mixture was stirred at room temperature for 24hours. After evaporating the solvent under reduced pressure, the residuewas purified by column chromatography to obtain the desired compound.

(8) Synthesis of Compound 9

To a 20 ml eggplant type flask, 0.25 g (0.41 mmol) of Compound 8 and 5.0ml of DMSO were added, and the mixture was stirred at room temperaturefor 2 hours. After evaporating the solvent under reduced pressure, theresidue was purified by column chromatography to obtain the desiredcompound.

EXAMPLE 6 Synthesis of Probe (No. 6)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and —CH₂CH₂NH₂ as R² was synthesized.

(1) Synthesis of phenyl-(2-trimethylsilanyloxy-ethoxy)-acetonitrile(Compound 2)

To a 30 ml eggplant type flask, 3.0 g (19.9 mmol) of2-phenyl-1,3-pentadione (Compound 1) was added and the flask was dippedin an ice bath. To the flask, 2.1 ml (21.43 mmol) of TMSCN and 0.3 g(0.94 mmol) of ZnI₂ were added, and the mixture was stirred at roomtemperature for 2 hours. Diethyl ether was added to the reactionsolution and the mixture was washed with water, followed by drying usingMgSO₄. The solvent was evaporated under reduced pressure to obtain ayellow oily product (4.17 g, yield: 83.2%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.15 (s, 9H, SiCH₃), 3.80 (t,2H, —OCH₂CH₂OSi—), 4.11 (t, 2H, —OCH₂CH₂OSi—), 5.39 (s, 1H, ArCH), 7.45(m, 5H, ArH)ESI-TOF(+): [M+Na]⁺=272.0

(2) Synthesis of 2-(2-amino-1-phenyl-ethoxy)-ethanol (Compound 3)

To a 100 ml three-necked flask, 1.0 g (4.01 mmol) of Compound 2 wasadded and the atmosphere was changed to nitrogen after degassing. Theflask was dipped in an ice bath and 30 ml of 1M BH₃ solution in THF wasslowly added. The resulting mixture was stirred for 30 minutes whilecooling the mixture in ice and then at room temperature for 4 hours.After completion of the reaction, the reaction vessel was dipped in anice bath and aqueous 1N HCl solution was added, thereby making themixture acidic. After evaporating the solvent under reduced pressure, 20ml of water was added and aqueous NaOH solution was added to adjust thepH to 10. The mixture was extracted with ethyl acetate and the organicphase was washed with water, followed by drying over anhydrous sodiumsulfate to obtain a colorless oily compound (650 mg, Yield: 89.5%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.95(d, 2H, NH₂—CH₂), 3.65(t,2H, —OCH₂CH₂OH), 3.75(t, 2H, —OCH₂CH₂OH), 4.46(t, 1H, ArCH), 7.32(m, 5H,ArH) ESI-TOF (+): [M+H]⁺=182.0

(3) Synthesis of [2-(2-hydroxy-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester

To a 100 ml two-necked flask, 1.40 g (7.70 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen. While cooling the flask in aice bath, 55 ml of anhydrous THF, 0.78 g (7.69 mmol) of TEA and 1.68 g(7.70 mmol) of di-tert-butyl-dicarbonate were added, and the mixture wasstirred at room temperature for 2 hours. The solvent was evaporatedunder reduced pressure and the residue was purified by columnchromatography (SiO₂, chloroform) to obtain a yellow oily compound (1.30g, Yield: 59.9%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.48(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₃), 3.49(t, 2H, —OCH₂CH₂OH), 3.73(t, 2H, —OCH₂CH₂OH) 4.42(t, 1H,ArCH), 7.33(m, 5H, ArH) ESI-TOF (+): [M+Na]⁺=304.0

(4) Synthesis of methanesulfonicacid-2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy) ethyl ester(Compound 5)

To a 50 ml eggplant type flask, 100 mg (0.28 mmol) of Compound 5 wasadded and the atmosphere was changed to nitrogen. Then 10 ml of acetoneand 1.0 g (6.67 mmol) of NaI were added, and the mixture was heated toreflux for 2 hours. After removing NaI by filtration, the solvent wasevaporated under reduced pressure. The residue was purified by columnchromatography (SiO₂, n-hexane:ethyl acetate=1:1 v/v) to obtain a yellowoily compound (104 mg, Yield: 93.99%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.45(s, 9H, t-Bu), 3.22(t, 2H,I—CH₂), 3.49(d, 2H, NH—CH₂), 3.67(t, 2H, —OCH₂), 4.43(t, 1H, ArCH),7.34(m, 5H, ArH)ESI-TOF (+): [M+Na]⁺=413.9

(5) Synthesis of [2-(2-iodo-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester (Compound 6)

To a 50 ml eggplant type flask, 100 mg (0.28 mmol) of Compound 5 wasadded and the atmosphere was changed to nitrogen. Then 10 ml of acetoneand 1.0 g (6.67 mmol) of NaI were added, and the mixture was heated toreflux for 2 hours. After removing NaI by filtration, the solvent wasevaporated under reduced pressure. The residue was purified by columnchromatography (SiO₂, n-hexane:ethyl acetate=1:1 v/v) to obtain a yellowoily compound (104 mg, Yield: 93.99%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.45(s, 9H, t-Bu), 3.22(t, 2H,I—CH₂), 3.49(d, 2H, NH—CH₂), 3.67(t, 2H, —OCH₂), 4.43(t, 1H, ArCH),7.34(m, 5H, ArH)ESI-TOF (+): [M+Na]⁺413.9

(6) Synthesis of[2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy)-ethyl]-triethyl-ammoniumiodide (Compound 7)

To a 50 ml eggplant type flask, 350 mg (0.90 mmol) of Compound 68.75 mlof toluene and 1.75 ml (17:33 mmol) of TEA were added, and the reactionwas carried out at 80° C. for 24 hours. After evaporating the solventunder reduced pressure, the residue was purified by large thin layerchromatography (SiO₂, chloroform:methanol=7:1 v/v) to obtain a reddishyellow solid product (383 mg, Yield: 66.70%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.42(t, 9H, CH₂—CH₃), 1:48(s,9H, t-Bu), 3.53(q, 6H, NR₃—CH₂), 3.70(t, 2H, —OCH₂CH₂N—), 3.72(d, 2H,NH—CH₂), 3.95(t, 2H, —OCH₂CH₂N—), 4.63(t, 1H, ArCH), 7.35(m, 5H, ArH)ESI-TOF (+): [M]⁺=365.2

(7) Synthesis of Compound 8

To a 30 ml eggplant type flask, 0.50 g (1.02 mmol) of Compound 7, 0.50ml of TFA and 10.0 ml of methylene chloride were added, and the mixturewas stirred at room temperature for 30 minutes. The solvent wasevaporated under-reduced pressure and the resultant was dried underreduced pressure using a pump. After changing the atmosphere tonitrogen, 20.0 ml of THF, 0.10 g (1.02 mmol) of TEA, 0.10 g (1.02 mmol)of BOP and 0.10 g (1.02 mmol) of O-alanine were added, and the mixturewas stirred at room temperature for 24 hours. After evaporating thesolvent under reduced pressure, the residue was purified by columnchromatography to obtain the desired compound.

EXAMPLE 7 Synthesis of Probe (No. 7)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and

as R² was synthesized.

(1) Synthesis of phenyl-(2-trimethylsilanyloxy-ethoxy)-acetonitrile(Compound 2)

To a 30 ml eggplant type flask, 3.0 g (19.9 mmol) of2-phenyl-1,3-pentadione (Compound 1) was added and the flask was dippedin an ice bath. To the flask, 2.1 ml (21.43 mmol) of TMSCN and 0.3 g(0.94 mmol) of ZnI₂ were added, and the mixture was stirred at roomtemperature for 2 hours. Diethyl ether was added to the reactionsolution and the mixture was washed with water, followed by drying usingMgSO₄. The solvent was evaporated under reduced pressure to obtain ayellow oily product (4.17 g, yield: 83.2%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 0.15(s, 9H, SiCH₃), 3.80(t,2H, —OCH₂CH₂OSi—), 4.11(t, 2H, —OCH₂CH₂OSi—), 5.39(s, 1H, ArCH), 7.45(m,5H, ArH)ESI-TOF(+): [M+Na]⁺=272.0

(2) Synthesis of 2-(2-amino-1-phenyl-ethoxy)-ethanol (Compound 3)

To a 100 ml three-necked flask, 1.0 g (4.01 mmol) of Compound 2 wasadded and the atmosphere was changed to nitrogen after degassing. Theflask was dipped in an ice bath and 30 ml of 1M BH₃ solution in THF wasslowly added. The resulting mixture was stirred for 30 minutes whilecooling the mixture in ice and then at room temperature for 4 hours.After completion of the reaction, the reaction vessel was dipped in anice bath and aqueous 1N HCl solution was added, thereby making themixture acidic. After evaporating the solvent under reduced pressure, 20ml of water was added and aqueous NaOH solution was added to adjust thepH to 10. The mixture was extracted with ethyl acetate and the organicphase was washed with water, followed by drying over anhydrous sodiumsulfate to obtain a colorless oily compound (650 mg, Yield: 89.5%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 2.95(d, 2H, NH₂—CH₂), 3.65(t,2H, —OCH₂CH₂OH), 3.75(t, 2H, —OCH₂CH₂OH), 4.46(t, 1H, ARCH), 7.32(m, 5H,ArH) ESI-TOF (+): [M+H]⁺182.0

(3) Synthesis of [2-(2-hydroxy-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester

To a 100 ml two-necked flask, 1.40 g (7.70 mmol) of Compound 3 was addedand the atmosphere was changed to nitrogen. While cooling the flask inan ice bath, 55 ml of anhydrous THF, 0.78 g (7.69 mmol) of TEA and 1.68g (7.70 mmol) of di-tert-butyl-dicarbonate were added, and the mixturewas stirred at room temperature for 2 hours. The solvent was evaporatedunder reduced pressure and the residue was purified by columnchromatography (SiO₂, chloroform) to obtain a yellow oily compound (1.30g, Yield: 59.9%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.48(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₃), 3.49(t, 2H, —OCH₂CH₂QH), 3.73(t, 2H, —OCH₂CH₂OH), 4.42(t, 1H,ArCH), 7.33(m, 5H, ArH) ESI-TOF (+): [M+Na]⁺=304.0

(4) Synthesis of methanesulfonic acid2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy) ethyl ester (Compound 5)

To a 50 ml eggplant type flask, 500 mg (1.78 mmol) of Compound 4 wasadded and the atmosphere was changed to nitrogen. Then 16 ml ofanhydrous THF and 0.5 ml (4.55 mmol) of TEA were added and the flask wasdipped in an ice bath. To the mixture, 440 mg (3.84 mmol) of MsCl wasadded and the resulting mixture was stirred at room temperature for 1hour. After evaporating the solvent under reduced pressure, chloroformwas added and the generated precipitates were removed by filtration,followed by concentrating the filtrate under reduced pressure. Theresulting product was purified by column chromatography (SiO₂, ethylacetate: n-hexane=2:1 v/v) to obtain a yellow oily product (491 mg,Yield: 77.0%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.44(s, 9H, t-Bu), 3.24(d, 2H,NH—CH₂), 3.44(t, 2H, —OCH₂CH₂OS—), 3.62(t, 2H, —OCH₂CH₂OS—), 4.34(t, 1H,ArCH), 7.33(m, 5H, ArH) ESI-TOF (+): [M+Na]⁺382.2

(5) Synthesis of [2-(2-iodo-ethoxy)-2-phenyl-ethyl]-carbamic acidtert-butyl ester (Compounds 6)

To a 50 ml eggplant type flask, 100 mg (0.28 mmol) of Compound 5 wasadded and the atmosphere was changed to nitrogen. Then 10 ml of acetoneand 1.0 g (6.67 mmol) of NaI were added, and the mixture was heated toreflux for 2 hours. After removing NaI by filtration, the solvent wasevaporated under reduced pressure. The residue was purified by columnchromatography (SiO₂, n-hexane:ethyl acetate=1:1 v/v) to obtain a yellowoily compound (104 mg, Yield: 93.99%).

¹H-NMR (270 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.45 (s, 9H, t-Bu), 3.22 (t,2H, I—CH₂), 3.49 (d, 2H, NH—CH₂), 3.67(t, 2H, —OCH₂), 4.43(t, 1H, ArCH),7.34(m, 5H, ArH)ESI-TOF (+): [M+Na]⁺=413.9

(6) Synthesis of[2-(2-tert-butoxycarbonylamino-1-phenyl-ethoxy)-ethyl]-triethyl-ammoniumiodide (Compound 7)

To a 50 ml eggplant type flask, 350 mg (0.90 mmol) of Compound 6, 8.75ml of toluene and 1.75 ml (17.33 mmol) of TEA were added, and thereaction was carried out at 80° C. for 24 hours. After evaporating thesolvent under reduced pressure, the residue was purified by large thinlayer chromatography (SiO₂, chloroform:methanol=7:1 v/v) to obtain areddish yellow solid product (383 mg, Yield: 66.70%).

¹H-NMR (300 MHz, CDCl₃, TMS, r.t., δ/ppm) 1.42(t, 9H, CH₂—CH₃), 1.48(s,9H, t-Bu), 3.53(q, 6H, NR₃—CH₂), 3.70(t, 2H, —OCH₂CH₂N—), 3.72(d, 2H,NH—CH₂), 3.95(t, 2H, —OCH₂CH₂N—), 4.63(t, 1H, ArCH), 7.35(m, 5H, ArH)ESI-TOF (+): [M]⁺=365.2

(7) Synthesis of Compound 8

To a 30 ml eggplant type flask, 0.50 g (1.02 mmol) of Compound 7, 0.50ml of TFA and 10.0 ml of methylene chloride were added, and the mixturewas stirred at room temperature for 30 minutes. The solvent wasevaporated under reduced pressure and the resultant was dried underreduced pressure using a pump. After changing the atmosphere tonitrogen, 20.0 ml of THF, 0.10 g (1.02 mmol) of TEA, 0.10 g (1.02 mmol)of BOP and 0.20 g (1.02 mmol) of 2-bromo-4-methylpentanoic acid wereadded, and the mixture was stirred at room temperature for 24 hours.After evaporating the solvent under reduced pressure, the residue waspurified by column chromatography to obtain the desired compound.

EXAMPLE 8 Synthesis of Probe (No. 8)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and cyclodextrin (the number of glucopyranose:7) as R² was synthesized.

(1) Synthesis of Compound 2

To a 100 ml eggplant type flask, 2.0 g (1.59 mmol) ofmono-6-deoxy-6-amino-β-cyclodextrin (1), 20.0 ml of acetic anhydride and10.0 ml of pyridine were added, and the mixture was stirred at roomtemperature for 24 hours. The reaction solution was poured into coldwater and the resulting mixture was extracted with ether. The resultingmixture was washed with saturated saline and dried over anhydrous sodiumsulfate, followed by evaporation of the solvent under reduced pressureto obtain the desired compound.

(2) Synthesis of Compound 3

To a 100 ml three-necked flask, 1.0 g (0.37 mmol) of Compound 2, 0.26 g(1.85 mmol) of methyl iodide, 0.05 g (0.40 mmol) of trimethylamine and20.0 ml of anhydrous DMF were added, and the mixture was stirred at roomtemperature for 24 hours under nitrogen gas flow. After evaporating thesolvent under reduced pressure, reprecipitation operation was carriedout to obtain the desired compound.

(3) Synthesis of Compound 4

To a 50 ml eggplant type flask, 1.0 g (0.35 mmol) of Compound 35.0 ml of1.0M aqueous NaOH solution and 20.0 ml of ethanol were added, and themixture was heated to reflux for 5 hours. After evaporating most of thesolvent under reduced pressure, 20 ml of water was added and the mixturewas acidified with 1N HCl. After evaporating the solvent under reducedpressure, reprecipitation operation was carried out to obtain thedesired compound.

EXAMPLE 9 Synthesis of Probe (No. 9)

In accordance with the scheme below, the above-described compound havinga quaternary amine as R¹ and —ONH₂ as R² was synthesized.

(1) Synthesis of Compound 2

To a 100 ml eggplant type flask, 3.0 g (27.5 mmol) of Compound 1, 30.0ml of water and 3.46 g (34.3 mmol) of triethylamine were added, and theflask was dipped in an ice bath. In 10.0 ml of diethyl ether, 4.8 g(27.5 mmol) of Z chloride was dissolved and the obtained solution wasadded to the mixture using a dropping funnel. The resulting mixture wasstirred for 30 minutes in an ice bath and then for 7 hours at roomtemperature. After extraction of the mixture with ethyl acetate, theorganic layer was washed with saturated saline. The resultant was driedover anhydrous sodium sulfate, and the residue was purified by columnchromatography (SiO₂, CHCl₃:ethyl acetate=2:3 v/v) to obtain the desiredcompound.

(2) Synthesis of Compound 3

To a 100 ml three-necked flask, 2.0 g (10.3 mmol of Compound 2, 50.0 mlof anhydrous methylene chloride and 1.1 g(11.0 mmol) of triethylaminewere added, and the flask was dipped in an ice bath. To the mixture, 1.4g (11.0 mmol) of p-xylylenediamine and 4.8 g (11.0 mmol) of BOP reagentwere added, and the resulting mixture was stirred in an iced bath for 30minutes and then at room temperature for 12 hours. After stopping thereaction by adding water, the mixture was washed with saturated salineand dried over anhydrous sodium sulfate. After evaporating the solventunder reduced pressure, the residue was purified by columnchromatography (SiO₂, CHCl₃:ethyl acetate=1:1 v/v) to obtain the desiredcompound.

(3) Synthesis of Compound 4

To a 100 ml eggplant type flask, 2.0 g (6.0 mmol) of Compound 3, 50.0 mlof toluene and 8.5 g (60.0 mmol) of methyl iodide were added, and themixture was heated at 80° C. under stirring. The generated precipitateswere recovered and washed with toluene to obtain the desired compound.

(4) Synthesis of Compound 5

To a 100 ml eggplant type flask, 2.0 g (5.0 mmol) of Compound 4 andethanol were added, and the atmosphere was changed to nitrogen, and thento hydrogen. To the mixture, 0.1 g of palladium-carbon was added and themixture was stirred at room temperature for 5 hours. After removing thepalladium-carbon by filtration, the solvent was evaporated under reducedpressure to obtain the desired compound.

EXAMPLE 10 Synthesis of Probe (No. 10)

In accordance with the scheme below the above-described compound havinga quaternary amine as R¹ and —NHNH₂ as R² was synthesized.

(1) Synthesis of Compound 2

To a 100 ml eggplant type flask, 3.0 g (19.8 mmol) of Compound 1, 50.0ml of acetone, 14.1 g (100.0 mmol) of methyl iodide and 6.9 g (50.0mmol) of potassium carbonate were added, and the mixture was heated toreflux for 24 hours under nitrogen gas flow. After removing potassiumcarbonate by filtration, the solvent was evaporated under reducedpressure. Chloroform was added to the reaction mixture and the generatedprecipitates were recovered, followed by washing the precipitates withchloroform to obtain the desired compound.

(2) Synthesis of Compound 3

To a 100 ml eggplant type flask, 2.0 g (6.25 mmol) of Compound 2, 30.0ml of ethanol and 0.22 g (7.0 mmol) of hydrazine were added, and themixture was stirred at room temperature for 5 hours. The solvent wasevaporated under reduced pressure to obtain the desired product.

EXAMPLE 11 Electrospray Ionization Mass Spectrometry

(1) Binding between Sample Compound and Probe

As shown in the reaction equation below, the probe (Compound 1)synthesized in Example 1 and a sample compound (Compound 2) were bound.This reaction was carried out by adding 10.0 mM of Compound 1 and asolution of Compound 2 in acetonitrile (or in THF) to a test tube,mixing the reactants, and by stirring the mixture at room temperaturefor 30 minutes.

(2) Electrospray Ionization Mass Spectrometry

After diluting the reaction product obtained in (1) to 1.0 μM,measurement was carried out using ESI-TOF mass spectrometer (Mariner)produced by Applied Biosystems. The constitution of the massspectrometer is shown in FIG. 3; From a syringe pump, continuouslymoving solvent (MeOH, water or the like) was streamed at a flow rate of10.0 μL/min. The sample solution was introduced from an injector using amicrosyringe. The sample solution moves to the mass spectrometer alongthe flow of the mobile solvent.

The set conditions of the mass spectrometer were as follows:

-   Spray tip potential: 3450 V-   Nozzle Potential: 184 V-   Quad RF voltage: 1000 V-   Flow rate of Nebulizer gas (N₂): 0.25 L/min.-   Flow rate of Auxiliary gas (N₂): 1.0 L/min.-   Temperature of the counter stream: 160° C.

The results of the measurement are shown in FIG. 1. For comparison, theprobe alone was subjected to the mass spectrometry after dilution to 1.0μM. The results are shown in FIG. 2.

As is apparent from FIGS. 1 and 2, the binding product between thesample compound and the probe exhibited a sharp peak at a positiondifferent from that obtained with the probe alone. By this, it wasproved that electrospray ionization mass spectrometry may be carried outwith high sensitivity and high accuracy by the method described above.

1. A method for mass spectrometry, comprising binding a probe for massspectrometry of liquid samples, which is represented by the Formula [I]:R²-A-R¹  [I] (wherein R¹ represents an ionic functional group whichbecomes an ion in a solvent, R² represents a structure which can bind toother substances, and A represents a spacer moiety) and further whereinsaid A is represented by the following Formula [III]:

(wherein R⁶ represents C₁-C₂₀ alkylene with the proviso that not lessthan one and not more than half of the —CH₂— units therein may besubstituted by one or more groups selected from the group consisting of—O—, —CO— and —NH—, and that said alkylene may be substituted by one ormore C₁-C₂₀ alkyl; and Ar represents an aromatic ring which may besubstituted by 1 to 5 C₁-C₂₀ alkyl) to a sample compound in a sampleliquid; and subjecting the obtained bound product to mass spectrometry.2. The method according to claim 1, wherein said R² is a functionalgroup which can react with said substance so as to covalently bind tosaid substance.
 3. The method according to claim 2, wherein said R¹ isan amine, carboxylic acid or a salt thereof, sulfonic acid or a saltthereof, or

(wherein R′, R″ and R′″ independently represent hydrogen, halogen orC₁-C₂₀ linear or branched alkyl).
 4. The method according to claim 3,wherein said R¹ is an amine represented by Formula [II]:

(wherein R³, R⁴ and R⁵ independently represent hydrogen, halogen orC₁-C₂₀ linear or branched alkyl).
 5. A method for mass spectrometry,comprising binding a probe for mass spectrometry of liquid samples,which is represented by the Formula [I]:R²-A-R¹  [I] (wherein R¹ represents an ionic functional group whichbecomes an ion in a solvent, R² represents a structure which can bind toother substance, and A represents a spacer moiety) to a sample compoundin a sample liquid; and subjecting the obtained bound product to massspectrometry, wherein said R² is

ClOC—, CH₃CH(NH₂)═CH—, —CH₂ONH₂—HCl, —NHNH₂,


6. The method according to claims 2 or 5, wherein said A has ahydrophobic moiety and a hydrophilic moiety.
 7. The method according toclaim 5, wherein said A is represented by the following Formula [III]:

(wherein R⁶ represents C ₁-C₂₀ alkylene with the proviso that not lessthan one and not more than half of the —CH₂— units therein may besubstituted by one or more groups selected from the group consisting of—O—, —CO—and —NH—, and that said alkylene may be substituted by one ormore C₁-C₂₀ alkyl; and Ar represents an aromatic ring which may besubstituted by 1 to 5 C₁-C₂₀ alkyl).
 8. The method according to claim 4,wherein said A is represented by the following Formula [IV]:

(wherein R⁷ may or may not exist, and when it exists, it representsC₁-C₆ alkylene; and R⁸ represents C₁-C₆ alkylene in which an arbitraryhydrogen is substituted by the benzene ring shown in Formula [IV]), orrepresented by the following Formula [V]:

(wherein R⁷ and R⁸ represent the same meanings as in Formula [IV]; R⁹may or may not exist, and when it exists, it represents C₁-C₆ alkylene).9. The method according to claims 2 or 5, which has a molecular weightof not more than
 1000. 10. The method according to claims 2 or 5, whichis for electrospray ionization mass spectrometry.
 11. A method for massspectrometry, comprising binding a probe for mass spectrometry of liquidsamples, which is represented by the Formula [I]:R²-A-R¹  [I] (wherein R¹ represents an ionic functional group whichbecomes an ion in a solvent, R² represents a structure which can bind toother substance, and A represents a spacer moiety) to a sample compoundin a sample liquid; and subjecting the obtained bound product to massspectrometry, wherein said R² is represented by Formula [VI]:

(wherein X represents halogen), or


12. The method according to claim 2, wherein said R² has a group havingoptical activity.
 13. The method according to claim 2, wherein said R²is represented by Formula [VIII]:

(wherein X represents halogen, and R¹⁰ represents C₁-C₅ alkyl).
 14. Themethod according to claim 1, wherein said R² has a structure whichintercalates into double-stranded nucleic acids.
 15. The methodaccording to claim 14, wherein said R² is represented by Formula [IX]:

(wherein R¹¹ and R¹² independently represent hydrogen, halogen, C₁-C₅alkyl or C₁-C₅ N,N-dialkylamino).
 16. The method according to claim 1,wherein said R² has a cyclic structure which can clathrate othersubstance.
 17. The method according to claim 16, wherein said R² isrepresented by Formula [X]:

(wherein R¹³ represents hydroxyl, carboxyl or C₁-C₅ alkyl; and mrepresents an integer of 5 to 9).
 18. The method according to claim 16,wherein said R² is represented by Formula [XI]:

(wherein R¹⁴ and R¹⁵ independently represent hydrogen, halogen or C₁-C₅alkyl; R¹⁶ represents C₁-C₅ alkyl or C₁-C₅ alkyl which has a carboxylgroup, ester group or an amide group at its terminal; and p representsan integer of 3 to 7).
 19. The method according to claim 11, whereinsaid R¹ is an carboxylic acid or a salt thereof, sulfonic acid or a saltthereof, or

(wherein R′, R″ and R′″ independently represent hydrogen, halogen orC₁-C₂₀ linear or branched alkyl).
 20. The method according to claim 19,wherein said R¹ is a amine represented by Formula [II]:

(wherein R³, R⁴ and R⁵ independently represent hydrogen, halogen orC₁-C₂₀ linear or branched alkyl).
 21. The method according to claim 11,wherein said A has a hydrophobic moiety and a hydrophilic moiety. 22.The method according to claim 4, wherein said A is represented by thefollowing Formula [III]:

(wherein R⁶ represents C₁-C₂₀ alkylene with the proviso that not lessthan one and not more than half of the —CH₂— units therein may besubstituted by one or more groups selected from the group consisting of—O—, —CO— and —NH—, and that said alkylene may be substituted by one ormore C₁-C₂₀ alkyl; and Ar represents an aromatic ring which may besubstituted by 1 to 5 C₁-C₂₀ alkyl).
 23. The method according to claim4, wherein said A is represented by the following Formula [IV]:

(wherein R⁷ may or may not exist, and when it exists, it representsC₁-C₆ alkylene; and R⁸ represents C₁-C₆ alkylene in which an arbitraryhydrogen is substituted by the benzene ring shown in Formula [IV]), orrepresented by the following Formula [V]:

(wherein R⁷ and R⁸ represent the same meanings as in Formula [IV]; R⁹may or may not exist, and when it exists, it represents C₁-C₆ alkyleneor phenylene).
 24. The method according to any one of claims 1, 5 or 11,wherein said A represents —R⁶— (wherein R⁶ represents the same meaningsas in Formula [III]) or —R⁶—Ar—R⁶′— wherein R⁶ and Ar represent the samemeanings as in Formula [III]; R^(6′) may or may not exist, and when itexists, it represents the same meanings as said R⁶ in Formula [III](with the proviso that R⁶ and R^(6′) in said formula may be the same ordifferent).
 25. The method according to claim 24, wherein said A isrepresented by the following Formula [XII]:

(wherein R¹⁷ and R¹⁸ independently represent C₁-C₆ alkylene; and R¹⁹ mayor may not exist, and when it exists, it represents C₁-C₆ alkylene);

or represented by the following Formula [XIII]: (wherein R²⁰ and R²¹independently represent C₁-C₆ alkylene; and R²² may or may not exist,and when it exists, it represents C₁-C₆ alkylene).
 26. The methodaccording to claim 11, which has a molecular weight of not more than1000.
 27. The method according to claim 11, which is for electrosprayionization mass spectrometry.